The above assays exploit the activity of various enzymes toward peroxides, according to the following general reactions: EQU Enzyme+ROOH.fwdarw.C+ROH (I) EQU C+AH.sub.2 .fwdarw.Enzyme+H.sub.2 O+A (II)
wherein C represents the enzyme-radical oxygen compound and A is a chromogen material which changes its color due to the loss of the hydrogen atoms.
The enzyme can be any enzyme which is active towards the peroxide, as hereinbefore defined, including but not limited to: peroxidase, catalase and myeloperoxidase. Throughout the following description, peroxidase will be used as the representative enzyme, for the sake of simplicity.
Examples of chromogenic materials are:
chloronaphthol, benzidine, tetramethylbenzidine, 3-amino-9-ethylcarbazole, dichloronaphthol, dibromonaphthol, 3,3',5,5'-tetramethylbenzidine dihydrochloride, 3,3',5,5'-tetramethylbenzidine (dihydrochloride dihydrate), 3-methylbenzothiazole-2,1-hydrazone, parahydroxyphenyl acetic acid, 2,2'-azino-di (3-ethylbenzothiazoline sulfonic acid), 4-aminoantipyrin/phenol, o-dianizidine, o-toluidine, 5-amino salicylic acid, guaiacol, o-tolidine, pyrogalol.
Examples of commonly employed assays are shortly described hereinafter:
Enzymeimmunoassay (EIA)
In this test, to a medium which is to be checked for the presence of a certain antigen there are added the appropriate insolubilized antibody namely, a peroxidase conjugated antibody. After washing, a substrate containing H.sub.2 O.sub.2 and a chromogen material is added. In the presence of the tested antigen, the peroxidase conjugated antibody is bound to the antigen. Addition of the substrate causes H.sub.2 O.sub.2 decomposition by peroxidase with evolution of color.
Immuno-Peroxidase Assay (IPA)
This method employs an antigen, an anti-antigen and a conjugate composed of peroxidase conjugated antibody anti-antibody anti-antigen. The peroxidase is bound and color evolves as above on addition of the substrate.
Peroxidase-anti-peroxidase Method (PAP)
This method commonly employs the reciprocal bonding of the following agents: antigen, anti-antigen (produced in rabbit), goat anti-rabbit IgG and peroxidase-anti-peroxidase (anti-peroxidase produced in rabbit). Binding of peroxidase is again obtained, leading to color development upon addition of the substrate.
In addition to the above assays, there are other extra-cellular tests which are customarily carried out, e.g., for detecting enzymatic activity in a given medium. In such cases, the substrate is directly added to the enzyme-containing medium and enzyme activity/concentration is obtained from the intensity of the color produced.
The above and similar assays are widely employed today for a variety of clinical tests. The use of these methods, however, is severely hampered by the need to prepare fresh substrate solutions shortly before use, since chromogen materials and peroxides cannot coexist for long periods of time, because of the spontaneous evolution of color which occurs due to the decomposition of the peroxide to give a an oxygen radical, which rapidly occurs under the influence of light, metal ions, impurities etc. A substrate which has spontaneously reacted in such a way cannot be employed to obtain reliable results and has to be replaced.
As it will be apparent to those skilled in the art, it would be highly desirable to be able to obtain a method for carrying out enzyme assays which employs substrates which can be stored prior to use without deteriorating, thereby simplifying and quickening all tests employing them.
In a patent application of the same applicant (namely, U.S. Ser. No. 771,417 filed Aug. 30, 1985), a method and chemical compositions for stabilizing mixtures of chromogen materials and peroxides and stable solutions obtained thereby are described.